Protein Antigen Production Overview

Selecting the proper antigen is one of the most critical steps in any custom antibody project. In most cases, scientists either use protein antigens or peptide antigens for this purpose. In some cases, an innovative new technique called DNA immunization allows the use of DNA as antigen. Among these choices, protein antigens are generally regarded as the gold standard when it comes to high-quality antigen production for antibody-generation purposes. Their unique ability to elicit antibodies that recognize the native confirmation of the target protein makes them a superior choice over other antigen alternatives. Given their structural complexity and size, proteins are inherently strong immunogens. Added to that, antibodies generated from protein antigens recognize multiple conformational epitopes within the target protein. This characteristic enhances antibody selectivity and specificity compared to anti-peptide antibodies. Selection of the ideal antigen production platform depends on a variety of factors, some of which are described ahead in this technical note.

When Choose a Protein Antigen?

You need an antibody that works in a variety of applications.

Your target protein has low sequence similarity to other proteins, is relatively easy to express and purify, and non-toxic.

Given GenScript Production Team's Experience and based on customer feedback we strongly recommend the use of Protein Antigen whenever possible, for the generation of high specificity antibodies.

Protein Antigen Production in E. coli

Recombinant E. coli protein expression is frequently used for the generation of protein antigens for antibody production. To generate high specificity antibodies against your target protein, there are several considerations that need to be looked at, before initiating recombinant expression.

Considerations for Protein Antigen Production in E. coli

Consideration Factor

Comments

Protein Solubility

An important consideration before embarking on antigen production in E. coli is to evaluate whether the protein of interest is known or predicted to be a soluble protein, peripheral membrane protein or an integral membrane protein.

Soluble proteins are relatively easier to work with since expression yields are better and purification becomes easier. Often times, in immunization protocols, soluble proteins are known to be easier to work with.

But despite the fact that soluble proteins make better protein antigens, insoluble proteins can be expressed in E. coli inclusion bodies, refolded and then used for immunization successfully.

Post Translational Modifications

Post translation modifications are important in protein antigen production because of their influence in protein functionality and localization.

For example, since many eukaryotic proteins are glycosylated, antigen production of such proteins in E. coli could adversely influence the reactivity of antibodies due to lack of such glycosylation.

If antibodies against antigens with certain kinds of modifications are required, other methods for antigen production will have to be pursued - for example, E.coli-produced antigens are not ideal for injecting humanized mice. CHO cells are a preferred alternative for such applications due to their better immunogenicity profile.

Subunit Structure

Oligomerization status of proteins is an important criteria for antigen production.

Oligomerization influences the ability to express the protein of interest as a soluble protein - a monomeric protein can be lot easier to produce than a multimeric protein.

In case of multimeric proteins, identifying a soluble domain fragment of the protein within the larger protein might be a better choice for protein antigen production.

Conserved Domains

Whether the antigen is a product of a conserved gene family or it is unique is an important consideration – in order to generate isoform-specific antibodies, expression of sequence divergent domains may be necessary.

Choosing a protein fragment that does not contain a highly conserved protein domain could aid in minimizing cross-reactivity of the antibody generated.

Schematic of Protein Antigen Production in E. coli at GenScript

Tags used for Protein Antigen Production in E. coli

To facilitate affinity purification and/or solubility, most antigens produced in bacteria are fused to a tag. Few commonly used tag choices are listed in the table below.

Tag

Features

His Tag

Usually polyhistidine tags such as 6-His, 8-His are used.

Recombinant tagged proteins are purified using affinity matrix such as Ni-NTA which have metal ions Ni or Co to which the His-tagged protein binds with micromolar affinity.

Elution is usually carried out using Imidazole.

Can be used under denaturing conditions.

Pros include small size and simplicity of use and cons include non-specific impurities during purification.

MBP Tag

~42kDa naturally occurring protein found in E. coli, encoded by the malE gene.

MBP can significantly enhance recombinant protein solubility and some suggest it could be an immunostimulant.

Elution is carried out, usually under non-denaturing conditions, using maltose.

Pros include enhanced recombinant protein solubility and cons include fragility of amylose resin and steric hindrance from MBP tag. For more information on the MBP tag, read the MBP tag technical note.

GST Tag

~25kDa tag first isolated from the parasite Schistosoma japonicum.

Recombinant tagged proteins are purified by binding to sepharose beads coated with glutathione.

Elution is carried out by addition of excess free-glutathione in solution.

Pros include a well-characterized system that works well and aids in improving recombinant protein solubility. Cons include GST-dimerization which could complicate downstream protein purification.

Small tags such as the 6His tag are less immunogenic but larger tags such as MBP and GST normally need to be removed from the antigen to minimize the incidental production of antibodies to the tag, in addition to the desired antigen. In case larger epitope tags are not cleaved prior to protein use as immunogen, additional counter screening may have to be performed in order to identify antibodies that bind uniquely to the protein antigen of interest.

Protein Antigen Production in Mammalian System

Mammalian antigen production is often preferred for the production of eukaryotic proteins. The advantage of a mammalian system is its ability to confer the appropriate post translational modifications, particularly, glycosylation. Combined with its efficient protein folding system, antigens produced in mammalian systems often resemble native conformation and demonstrate better solubility. Mammalian antigen production is therefore the system of choice for many proteins that are either eukaryotic, difficult to produce or required for antibody generation projects where detection of native antigen is absolutely critical. GenScript provides high quality transient expression services that are suitable for the production of your antigens in either CHO or HEK293 cells. Visit MamPower™ Guaranteed Mammalian Protein Expression page to learn more.

Recommended Protein Antigen Production Services at GenScript

With years of experience in developing customized Antibodies against difficult targets, GenScript brings the unique perspective of antibody experts to recombinant protein antigen production. While E. coli expression is the most routinely adopted method for protein antigen production, in some instances mammalian expression may be necessary.

Moving directly to antibody generation? GenScript seamlessly integrates protein production with antibody production services. We provide fast and guaranteed results with our PolyExpress™ Premium and MonoExpress™ Premium packages. Your project can progress from protein sequence to validated antibodies in as little as 18 weeks, including protein synthesis. Get a quote today to accelerate your research!

Quotations and Ordering

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